Epstein-Barr virus-induced gene 2 (EBI2) (1-4), also known as GPR183, is expressed on B cells and is highly induced upon activation (5). Recent gene targeting experiments revealed that EBI2-/- B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses (1, 2). Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2 (3, 4). Although other closely related oxysterols showed some potency and binding capability to EBI2, the most potent endogenous EBI2 receptor ligand and activator was 7alpha,25-dihydroxycholesterol (7alpha,25-OHC) (4). EBI2-/- B cells did not bind 7alpha,25-OHC and mice deficient for cholesterol 25-hydroxylase that is necessary to generate 7alpha,25-OHC display a phenotype similar to that of EBI2-/- mice (3). The recent publication in July 2011 of the deorphanizing of this receptor is a major step forward towards understanding the role of EBI2 in immunobiology. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective potent chemical probes that antagonize this receptor need to be identified and made freely available to the research community.

This cell-based luminescent assay is similar to "uHTS identification of small molecule antagonists of the EBI2 receptor via a luminescent beta-arrestin assay", AID 651636, and is performed for single-concentration reconfirmation of hits identified in primary screening.

Compounds that demonstrated a % activity of >=50% are defined as active compounds.To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Activity ScoringActivity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay and is not applicable:a. If outcome of the primary screen is inactive, then the assigned score is 0b. If outcome of the primary screen is inconclusive, then the assigned score is 10c. If outcome of the primary screen is active, then the assigned score is 20Scoring for Single concentration confirmation screening:d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay